Advances in Design and Development of Lumi-Solve: A Novel Drug-Eluting Photo-Angioplasty Device.

PURPOSE: The Lumi-Solve photo-angioplasty drug eluting balloon catheter (DEBc) may afford safety advantages over current DEBc. Lumi-Solve utilises the guidewire (GW) port and lumen to deliver fibre-optic UV365nm light to the angioplasty balloon which may be problematic. We explore and evaluate alternative Lumi-Solve design options to circumvent fibre-optic use of the GW port and lumen which may enhance efficacy and clinical utility. METHODS: Effects of guidewire shadowing (GWS) on visible and UV365nm light transmission were evaluated and modelled in-silico. To evaluate the effect of a dedicated intra-balloon fibre-optic port, modified angioplasty balloons and sections of translucent polyethylene terephthalate (PET) GW port tubing were utilised. Investigation of the effect of GWS on chemical and biological photo-activation of balloon surface drug was performed utilising LCMS analysis and inhibition of histone deacetylase activity (HDACi) was measured in human umbilical vein endothelial cells (HUVEC). RESULTS: Parallel fibre-optic and GW port configurations generated a GWS of approximately 18.0% of the evaluable balloon surface area and attenuated both visible and UV light intensity by 20.0-25.0% and reduced chemical photo-activation of balloon surface drug and HDACi by at least 40-45%. Alternative fibre-optic port configurations including a spiral design significantly mitigated GWS effects on UV light transmission. CONCLUSIONS: To avoid use of the GW port and its associated complications a dedicated third port and lumen for the Lumi-Solve fibre-optic may be required. To maximize balloon surface chemical and biological photo-activation, non-parallel, intra-balloon, fibre-optic lumen trajectories, including a spiral design may be useful.

Nerve guidance conduit development for primary treatment of peripheral nerve transection injuries: A commercial perspective.

Commercial nerve guidance conduits (NGCs) for repair of peripheral nerve discontinuities are of little use in gaps larger than 30 mm, and for smaller gaps they often fail to compete with the autografts that they are designed to replace. While recent research to develop new technologies for use in NGCs has produced many advanced designs with seemingly positive functional outcomes in animal models, these advances have not been translated into viable clinical products. While there have been many detailed reviews of the technologies available for creating NGCs, none of these have focussed on the requirements of the commercialisation process which are vital to ensure the translation of a technology from bench to clinic. Consideration of the factors essential for commercial viability, including regulatory clearance, reimbursement processes, manufacturability and scale up, and quality management early in the design process is vital in giving new technologies the best chance at achieving real-world impact. Here we have attempted to summarise the major components to consider during the development of emerging NGC technologies as a guide for those looking to develop new technology in this domain. We also examine a selection of the latest academic developments from the viewpoint of clinical translation, and discuss areas where we believe further work would be most likely to bring new NGC technologies to the clinic. STATEMENT OF SIGNIFICANCE: NGCs for peripheral nerve repairs represent an adaptable foundation with potential to incorporate modifications to improve nerve regeneration outcomes. In this review we outline the regulatory processes that functionally distinct NGCs may need to address and explore new modifications and the complications that may need to be addressed during the translation process from bench to clinic.

Design, Development, In Vitro and Preliminary In Vivo Evaluation of a Novel Photo-Angioplasty Device: Lumi-Solve.

PURPOSE: Paclitaxel (PTX)-coated drug eluting balloon catheters (DEBc) used in the management of neointimal hyperplasia (NIH) have been associated with safety concerns. Alternative coating agents and targeted delivery systems may improve safety and DEBc efficacy. Utilizing a multi-platform approach we designed, developed and evaluated Lumi-Solve, a novel DEBc, coated with ultraviolet (UV) 365 nm-activated caged metacept-3 (c-MCT-3), an epigenetic agent from the histone deacetylase inhibitor (HDACi) class. METHODS: In vitro catheter and contrast media transmission of UV365nm was evaluated spectroscopically. UV365nm conversion of c-MCT-3 to MCT-3 was evaluated chromatographically. Cellular toxicity and HDACi activity of c-MCT-3 ∓UV365nm was evaluated in vitro. In vivo UV365nm conversion of c-MCT-3 to MCT-3 was evaluated in an ovine carotid artery model. RESULTS: Catheter material and dilute contrast media did not attenuate UV365nm transmission or c-MCT-3 activation. c-MCT-3 demonstrated less cellular toxicity than MCT-3 and PTX. UV365nm-activated c-MCT-3 demonstrated HDACi activity. In vivo activation of c-MCT-3 produced MCT-3. CONCLUSIONS: Lumi-Solve, a novel DEBc device developed utilizing a combination of chemical, fibre-optic and catheter based technology platforms, demonstrated potential for targeted delivery of bioactive HDACi to the blood vessel wall supporting direct application to the management of NIH and warranting additional in vivo studies.

Extending Circulating Tumor DNA Analysis to Ultralow Abundance Mutations: Techniques and Challenges.

Liquid biopsies that analyze circulating tumor DNA (ctDNA) hold great promise in the guidance of clinical treatment for various cancers. However, the innate characteristics of ctDNA make it a difficult target: ctDNA is highly fragmented, and found at very low concentrations, both in absolute terms and relative to wildtype species. Clinically relevant target sequences often differ from the wildtype species by a single DNA base pair. These characteristics make analyzing mutant ctDNA a uniquely difficult process. Despite this, techniques have recently emerged for analyzing ctDNA, and have been used in pilot studies that showed promising results. These techniques each have various drawbacks, either in their analytical capabilities or in practical considerations, which restrict their application to many clinical situations. Many of the most promising potential applications of ctDNA require assay characteristics that are not currently available, and new techniques with these properties could have benefits in companion diagnostics, monitoring response to treatment and early detection. Here we review the current state of the art in ctDNA detection, with critical comparison of the analytical techniques themselves. We also examine the improvements required to expand ctDNA diagnostics to more advanced applications and discuss the most likely pathways for these improvements.

Increased Cardiomyocyte Alignment and Intracellular Calcium Transients Using Micropatterned and Drug-Releasing Poly(Glycerol Sebacate) Elastomers.

Myocardial tissue engineering is a promising therapy for myocardial infarction recovery. The success of myocardial tissue engineering is likely to rely on the combination of cardiomyocytes, prosurvival regulatory signals, and a flexible biomaterial structure that can deliver them. In this study, poly(glycerol sebacate) (PGS), which exhibits stable elasticity under repeated tensile loading, was engineered to provide physical features that aligned cardiomyocytes in a similar manner to that seen in native cardiac tissue. In addition, a small molecule mimetic of brain derived neurotrophic factor (BDNF) was polymerized into the PGS to achieve a continuous and steady release. Micropatterning of PGS elastomers increased cell alignment, calcium transient homogeneity, and cell connectivity. The intensity of the calcium transients in cardiomyocytes was enhanced when cultured on PGS which released a small molecule BDNF mimetic. This study demonstrates that robust micropatterned elastomer films are a potential candidate for the delivery of functional cardiomyocytes and factors to the injured or dysfunctional myocardium, as well as providing novel in vitro platforms to study cardiomyocyte physiology.

Controlling integrin-based adhesion to a degradable electrospun fibre scaffold via SI-ATRP.

While polycaprolactone (PCL) and similar polyesters are commonly used as degradable scaffold materials in tissue engineering and related applications, non-specific adsorption of environmental proteins typically precludes any control over the signalling pathways that are activated during cell adhesion to these materials. Here we describe the preparation of PCL-based fibres that facilitate cell adhesion through well-defined pathways while preventing adhesion via adsorbed proteins. Surface-initiated atom transfer radical polymerisation (SI-ATRP) was used to graft a protein-resistant polymer brush coating from the surface of fibres, which had been electrospun from a brominated PCL macroinitiator. This coating also provided alkyne functional groups for the attachment of specific signalling molecules via the copper-mediated azide-alkyne click reaction; in this case, a cyclic RGD peptide with high affinity for αvß3 integrins. Mesenchymal stem cells were shown to attach to the fibres via the peptide, but did not attach in its absence, nor when blocked with soluble peptide, demonstrating the effective control of cell adhesion pathways.

Low Fouling Electrospun Scaffolds with Clicked Bioactive Peptides for Specific Cell Attachment.

While electrospun fibers are of interest as scaffolds for tissue engineering applications, nonspecific surface interactions such as protein adsorption often prevent researchers from controlling the exact interactions between cells and the underlying material. In this study we prepared electrospun fibers from a polystyrene-based macroinitiator, which were then grafted with polymer brushes using surface-initiated atom transfer radical polymerization (SI-ATRP). These brush coatings incorporated a trimethylsilyl-protected PEG-alkyne monomer, allowing azide functional molecules to be covalently attached, while simultaneously reducing nonspecific protein adsorption on the fibers. Cells were able to attach and spread on fibrous substrates functionalized with a pendant RGD-containing peptide, while spreading was significantly reduced on nonfunctionalized fibers and those with the equivalent RGE control peptide. This effect was observed both in the presence and absence of serum in the culture media, indicating that protein adsorption on the fibers was minimal and cell adhesion within the fibrous scaffold was mediated almost entirely through the cell-adhesive RGD-containing peptide.

Optimization of Aqueous SI-ATRP Grafting of Poly(Oligo(Ethylene Glycol) Methacrylate) Brushes from Benzyl Chloride Macroinitiator Surfaces.

Poly(oligo(ethylene glycol) methacrylate) (pOEGMA) brushes were grafted via surface-initiated atom transfer radical polymerization (SI-ATRP) from a poly(styrene-co-vinylbenzyl chloride) macroinitiator. While bromoisobutyryl initiator groups are most commonly used for this purpose, benzyl chloride initiators may be advantageous for some applications due to superior stability. Water-only graft solutions produced thicker brush coatings with superior low fouling properties (low protein adsorption and cell adhesion) versus mixed water/alcohol solutions. Coatings produced using 475 Da OEGMA (methyl ether terminated) further reduced non-specific interactions compared to 360 Da OEGMA (hydroxyl terminated). Initiator density had minimal effect on low fouling properties.

Mitochondrial DNA haplotypes define gene expression patterns in pluripotent and differentiating embryonic stem cells.

Mitochondrial DNA haplotypes are associated with various phenotypes, such as altered susceptibility to disease, environmental adaptations, and aging. Accumulating evidence suggests that mitochondrial DNA is essential for cell differentiation and the cell phenotype. However, the effects of different mitochondrial DNA haplotypes on differentiation and development remain to be determined. Using embryonic stem cell lines possessing the same Mus musculus chromosomes but harboring one of Mus musculus, Mus spretus, or Mus terricolor mitochondrial DNA haplotypes, we have determined the effects of different mitochondrial DNA haplotypes on chromosomal gene expression, differentiation, and mitochondrial metabolism. In undifferentiated and differentiating embryonic stem cells, we observed mitochondrial DNA haplotype-specific expression of genes involved in pluripotency, differentiation, mitochondrial energy metabolism, and DNA methylation. These mitochondrial DNA haplotypes also influenced the potential of embryonic stem cells to produce spontaneously beating cardiomyocytes. The differences in gene expression patterns and cardiomyocyte production were independent of ATP content, oxygen consumption, and respiratory capacity, which until now have been considered to be the primary roles of mitochondrial DNA. Differentiation of embryonic stem cells harboring the different mitochondrial DNA haplotypes in a 3D environment significantly increased chromosomal gene expression for all haplotypes during differentiation. However, haplotype-specific differences in gene expression patterns were maintained in this environment. Taken together, these results provide significant insight into the phenotypic consequences of mitochondrial DNA haplotypes and demonstrate their influence on differentiation and development. We propose that mitochondrial DNA haplotypes play a pivotal role in the process of differentiation and mediate the fate of the cell.

Surface grafting of electrospun fibers using ATRP and RAFT for the control of biointerfacial interactions.

BACKGROUND: The ability to present signalling molecules within a low fouling 3D environment that mimics the extracellular matrix is an important goal for a range of biomedical applications, both in vitro and in vivo. Cell responses can be triggered by non-specific protein interactions occurring on the surface of a biomaterial, which is an undesirable process when studying specific receptor-ligand interactions. It is therefore useful to present specific ligands of interest to cell surface receptors in a 3D environment that minimizes non-specific interactions with biomolecules, such as proteins. METHOD: In this study, surface-initiated atom transfer radical polymerization (SI-ATRP) of poly(ethylene glycol)-based monomers was carried out from the surface of electrospun fibers composed of a styrene/vinylbenzyl chloride copolymer. Surface initiated radical addition-fragmentation chain transfer (SI-RAFT) polymerisation was also carried out to generate bottle brush copolymer coatings consisting of poly(acrylic acid) and poly(acrylamide). These were grown from surface trithiocarbonate groups generated from the chloromethyl styrene moieties existing in the original synthesised polymer. XPS was used to characterise the surface composition of the fibers after grafting and after coupling with fluorine functional XPS labels. RESULTS: Bottle brush type coatings were able to be produced by ATRP which consisted of poly(ethylene glycol) methacrylate and a terminal alkyne-functionalised monomer. The ATRP coatings showed reduced non-specific protein adsorption, as a result of effective PEG incorporation and pendant alkynes groups existing as part of the brushes allowed for further conjugation of via azide-alkyne Huisgen 1,3-dipolar cycloaddition. In the case of RAFT, carboxylic acid moieties were effectively coupled to an amine label via amide bond formation. In each case XPS analysis demonstrated that covalent immobilisation had effectively taken place. CONCLUSION: Overall, the studies presented an effective platform for the preparation of 3D scaffolds which contain effective conjugation sites for attachment of specific bioactive signals of interest, as well as actively reducing non-specific protein interactions.

Implantation of functionalized thermally gelling xyloglucan hydrogel within the brain: associated neurite infiltration and inflammatory response.

To develop neural tissue engineering strategies that are useful for repairing damaged neural pathways in the central nervous system, it is essential to control and optimise neurone and neurite interactions with functional scaffolds. In this study, the suitability of thermally gelling xyloglucan hydrogels, along with xyloglucan-graft-poly-D-lysine (PDL) hydrogels, was assessed through their implantation within the caudate putamen of adult rats. The ability of the hydrogel scaffolds to encourage the infiltration of axons in a controlled manner was investigated, as was the inflammatory response associated with the implantation. The microglia reaction was the same for unmodified xyloglucan and the xyloglucan-graft-PDL scaffolds, peaking after 3 days before decreasing back to homeostatic levels after approximately 28 days. Penetration of the microglia into the scaffold was not observed, with these cells accumulating at the scaffold-tissue interface. For astrocytes, the other type of glial cell with migratory capacity, the peak activation occurred between 14 and 21 days. This reaction subsided more rapidly for the unmodified scaffold compared to the xyloglucan-graft-PDL scaffolds, which remained elevated 21-28 days before returning to homeostatic levels within 60 days. Most noteworthy was the discovery of increased infiltration levels for astrocytes and neurites with higher concentrations of grafted PDL. The timing of the astrocyte migration coincided with neurite infiltration within the scaffolds, suggesting that astrocytes may have facilitated this infiltration, possibly due to the secretion of laminin.

Neurite infiltration and cellular response to electrospun polycaprolactone scaffolds implanted into the brain.

Assessment of axonal infiltration and guidance within neural tissue engineering scaffolds, along with the characterisation of the inflammatory response, is critical in determining these scaffolds' potential for facilitating neural repair. In this study, the extent of microglial and astrocytic response was measured following implantation of electrospun poly(epsilon-caprolactone) (PCL) scaffolds into the caudate putamen of the adult rat brain. The inflammation peaked at around 4 days (microglia) and 7 days (astrocytes) and subsided to homeostatic levels by 60 days. There was no evidence of microglial encapsulation and indeed neurites had infiltrated the implants, evidence of scaffold-neural integration. Whilst the inflammatory response was uninfluenced by the degree of PCL fibre alignment, the extent of neurite entry was. Large porosity, as was the case with the randomly orientated polymer fibres, enabled neurite infiltration and growth within the scaffold. However, neuronal processes could not penetrate scaffolds when fibres were partially aligned and instead, preferentially grew perpendicular to the direction of PCL fibre alignment at the implant-tissue interface i.e. perpendicular, not parallel, contact guidance was provided. This investigation shows that electrospun PCL fibres are compatible with brain tissue and provide preliminary insights regarding the influence of microglia and astrocytes in neural integration within such scaffolds.

Surface and bulk characterisation of electrospun membranes: problems and improvements.

Electrospun membranes are used in a variety of applications, including filtration systems and sensors for chemical detection, and have attracted increased interest in the field of tissue engineering and regenerative medicine. Successful integration of these materials into a specific technology will require understanding of the fibres' surface, bulk and architectural properties. Detailed characterisation of these properties is frequently overlooked, particularly in specialised interdisciplinary fields such as tissue engineering. In this article we have reviewed the current status of the characterisation of electrospun membranes, while recommending improvements in using these techniques to better understand these very interesting nanostructured materials.